POS0430 THE EXPANDED CD21LOW B CELL SUBPOPULATION IN ANKYLOSING SPONDYLITIS CONSISTS MAINLY OF UNMUTATED ANTIGEN-INEXPERIENCED CELLS THAT ARE CLONALLY RELATED TO MEMORY B CELLS AND PLASMABLASTS
Authors: R. Wilbrink, L. Van der Weele, A. Spoorenberg, N. De Vries, F. G. M. Kroese, G. M. Verstappen
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> The role of B cells in the pathogenesis of ankylosing spondylitis (AS) remains relatively understudied. Nevertheless, available evidence shows presence of B cells at sites of inflammation, higher frequencies of circulating plasmablasts, presence of autoantibodies and beneficial results of B cell depleting therapy with rituximab [1]. We observed previously that a subpopulation of CD27<sup>–</sup> B cells, characterized by low expression of the complement receptor CD21 (CD21<sup>low</sup>), is increased in patients with AS and patients with primary Sjogren's syndrome (pSS), a typical B cell mediated systemic autoimmune disease [2]. These CD21<sup>low</sup>CD38<sup>low</sup>CD27<sup>–</sup> B cells display a pro-inflammatory phenotype, suggestive for a pathogenic role. <h3>Objectives</h3> To gain insight into the origin of these CD21<sup>low</sup>CD38<sup>low</sup>CD27<sup>–</sup> B cells we analysed the mutation status of the immunoglobulin heavy chain variable (IGHV) gene regions in the total B cell compartment, CD21<sup>low</sup> B cell subpopulations and plasmablasts in AS patients in comparison to pSS patients and healthy donors (HD). In addition, we investigated clonal relations between CD21<sup>low</sup> B cell subpopulations and plasmablasts in AS. <h3>Methods</h3> RNA was isolated from sorted total B cells (CD19<sup>+</sup>), two CD21<sup>low</sup> B cell populations (CD21<sup>low</sup>CD38<sup>low</sup>CD27<sup>–</sup> and CD21<sup>low</sup>CD38<sup>low</sup>CD27<sup>+</sup>) and plasmablasts (CD19<sup>+</sup>CD38<sup>++</sup>CD27<sup>+</sup>) from peripheral mononuclear cells of 10 AS patients fulfilling the mNY criteria (mean age 46 ± 9 years, 60% male, mean ASDAS 2.3 ± 0.8, all HLA-B27+), 10 sex- and age-matched HDs (mean age 48 ± 21 years, 60% male) and 10 age-matched pSS patients (mean age 46 ± 9 years, 20% male). Repertoire analysis of the B cell receptor heavy chain was performed using high throughput sequencing on the Illumina MiSeq Platform. Sequences were analyzed for degree of somatic hypermutation, usage of immunoglobulin heavy chain variable (IGHV) and joining (IGHJ) regions and clonal relationships. <h3>Results</h3> IGHV sequences with identical heavy chain CDR3 regions were grouped together and were defined as a clone. Analysis of the IGHV mutation status in CD21<sup>low</sup> B cells revealed that the majority of clones (71% SD ± 14.1) in the CD21<sup>low</sup>CD38<sup>low</sup>CD27<sup>–</sup> subset from patients with AS consisted of clones with a low mutation rate (0-2 mutations in the IGHV region) (Figure 1b). On the contrary, most of the clones in the CD21<sup>low</sup>CD38<sup>low</sup>CD27<sup>+</sup> (49% SD ± 12.5) and plasmablasts (59% SD ± 12.7) subsets were clones with a higher mutation load (&gt;10 mutations in the IGHV region). A similar mutational pattern was seen in pSS patients and HDs. In AS, IGHV3 and IGHJ4 family genes were predominantly used (Figure 1c). However, we observed no differences in family gene usage among B cell subsets nor between groups. To investigate clonal relationships we performed a CDR3 amino acid network analysis on the top 100 most expanded clones per subset in AS patients and found that the CD21<sup>low</sup>CD38<sup>low</sup>CD27<sup>–</sup> subset shared more clones with the CD21<sup>low</sup>CD38<sup>low</sup>CD27<sup>+</sup> subset (median 3.0 (0.8-7.0 IQR), p&lt; 0.05) compared to plasmablasts (median 0.0 (0-1.3 IQR). <h3>Conclusion</h3> This study shows that the expanded circulating CD21<sup>low</sup>CD38<sup>low</sup>CD27<sup>–</sup> B cell population in AS consists mainly of antigen-inexperienced cells. The presence of shared clones with their CD27<sup>+</sup> counterpart and plasmablasts suggest that the three subsets are to some extent related. Hence, CD21<sup>low</sup>CD38<sup>low</sup>CD27<sup>–</sup> B cells may differentiate towards CD21<sup>low</sup>CD38<sup>low</sup>CD27<sup>+</sup> B cells and plasmablasts. The relative roles of these CD21<sup>low</sup> B cell subsets and plasmablasts in AS pathogenesis remain to be established. <h3>References</h3> [1]Wilbrink, R et al. (2021). B Cell Involvement in the Pathogenesis of Ankylosing Spondylitis. <i>International journal of molecular sciences</i>. [2]Wilbrink, R et al. (2021). CD27<sup>-</sup>CD38<sup>low</sup>CD21<sup>low</sup> B-Cells Are Increased in Axial Spondyloarthritis. <i>Frontiers in immunology</i>. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> Rick Wilbrink: None declared, Linda Van der weele: None declared, Anneke Spoorenberg Consultant of: Abbvie, Pfizer, MSD, UCB, Lilly and Novartis, Grant/research support from: Abbvie, Pfizer, Union Chimique Belge (UCB), Novartis, Niek de Vries: None declared, Frans G.M. Kroese Speakers bureau: BMS Roche and Jannsen-Cilag, Consultant of: BMS, Grant/research support from: BMS, Gwenny M. Verstappen: None declared.